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International Journal of Medical Science and Innovative Research (IJMSIR)
IJMSIR : A Medical Publication Hub
Available Online at: www.ijmsir.com
Volume – 3, Issue –3, June - 2018, Page No. : 196 – 211
Corresponding Author: Rubaba Karim, Volume – 3 Issue - 3, Page No. 196 - 211
Page 196
ISSN- O: 2458 - 868X, ISSN–P: 2458 – 8687
Index Copernicus Value: 49. 23
SJIF Impact Factor: 4.271
Evaluation of Antiulcerogenic Activity of Bacopa Monnieri (LINN.) on Ethanol-Induced Gastric Injury In Mice
Rubaba Karim1 , Ashfia Fatima Khan1, Sabiha Akter1, Najia Sultana Shuchi1, Mst. Maliha Tabachasum Madhuri1, Md.
Nazmul Hossain1
1Department of Pharmacy, Primeasia University, Dhaka, Bangladesh
Correspondence Author: Rubaba Karim, Lecturer, Department of Pharmacy, Primeasia University, Dhaka – 1213,
Bangladesh
Type of Publication: Original Research Paper
Conflicts of Interest: Nil
Abstract
Bacopa monnieri (L) is a renowned medicinal plant for its
numerous therapeutic activities against a number of
ailments. Bangladeshi species of B. monnieri has been
previously reported for its anti-inflammatory activities.
Therefore, the present investigation involves
phytochemical screening and evaluation of
antiulcerogenic activity of B. monnieri extracts of
different solvent medium by in-vivo analysis. The study
was conducted by using ethanol-induced gastric injury in
Swiss albino mice model to evaluate anti-ulcer effect of B.
monnieri (whole plant). Three different solvent systems
(water, ethanol and carbon tetrachloride) were used to
prepare plant extract. Animals were pretreated with
200mg/kg and 400mg/kg (body weight, p.o.) of the plant
extracts in 0.2% tween 80 where omeprazole (20 mg/kg
b.w. in 0.2% tween 80, p.o.) was used as the reference
drug. The extent of gastric injury was evaluated by
assessment of gross appearance of gastric areas to
determine ulcer index and histology of stomach. A dose-
dependent therapeutic effect against gastric ulcer was
found with all three extracts of Bacopa monnieri where
ethanolic extract of the plant showed statistically
significant result(**p< 0.01) when compared with
ulcerogenic animal group. Furthermore, the therapeutic
effect of 400mg/kg (b.w. p.o.) of B. monnieri ethanolic
extract exhibited close relation with pretreatment by
omeprazole. The present study suggests ethanol as a
suitable extraction medium for Bacopa monnieri (L) in
order to evaluate its antiulcerogenic activity. Treatment
with this extract leads to extensive protection against
gastric ulcer providing a scientific basis for its traditional
uses as a cure for ulcer.
Keywords: Bacopa monnieri, antiulcerogenic, ulcer,
gastric injury, gastric ulcer
1. Introduction
Crude drugs of plant origin have been employed as
preventives and cure of different ailments for many years.
The interest in the employment of herbal products has
been growing day by day all over the world [1]. Bacopa
monnieri Linn. (Family: Scrophulariaceae), also known as
brahmi, bacopa and thyme leaved gratiola, has been
regarded as one of the popular herbal remedy in Ayurveda
system [2,3]. The plant is distributed abundantly in India,
Nepal, Srilanka, China, Taiwan, Vietnam, Florida and also
in many other warmer regions of the world [4].
Many phytochemical constituents have been isolated from
this plant that include alkaloid brahmine, nicotinine,
herpestine, bacosides A[3-( α-L-arabinopyranosyl)-O-β-
Dglucopyranoside-10,20-dihydroxy-16-keto-dammar-24-
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ene], triterpenoidsaponins , saponins A, B and C,
betulinic acid, D-mannitol, stigmastanol, β-sitosterol,
stigmasterol and pseudojujubogenin glycoside [5-12].
Bacopamonnieri extract has been reported to exhibit
several pharmacological activities, for example,
neuroprotective effect [13-15], an antioxidant [16-18], an
antiparkinsonian agent [19], hepatoprotective agent [20],
anti-depressant [21], anti-stress [22], anti-cancer [23],
antimicrobial effect [24,25], endocrine effect [26,27], anti-
inflammatory agent [28], gastrointestinal effect [29] and
antiulcerogenic agent [30].
Gastric ulcer is considered one of the most significant
gastrointestinal disorders that has become an alarming
health issue all over the world. Such ulcer occurs due to a
condition involving a lesion in the gastric mucosa that
perforates deep into the submucosal zone of the stomach
[31]. One of the major causes of this disorder is the
imbalanced function of defensive (mucus secretion,
cellular regeneration, prostaglandin, bicarbonate, nitric
oxide) and noxious (gastric acid and pepsin secretion)
factors of the gastrointestinal system [32]. This damages
the mucosal barrier resulting ulcer, inflamed cells and in
severe cases subepithelial hemorrhages in stomach
[33,34]. However, other factors that raises the incidence of
gastric ulcer involve smoking, stress, infection with
Helicobacter pylori, excessive or prolong consumption of
alcohol and prolong administration of NSAIDs [35-38].
Alcohol-induced gastric mucosal lesions is responsible for
developing gastritis, gastric ulcer and sometimes gastric
carcinoma [39]. Different experimental ulcer-induced
animal models have been reported in the previous studies
that involved ethanol and acetic acid-induced gastric
injury [40]. Alcohol damages the gastric mucosal layer in
the stomach, as a result, impairs mucus secretion (a
defensive factor) causing gastrointestinal erosions and
peptic ulcers [41-43]. This may even lead to more serious
complications, such as over bleeding or perforation [44,
45].
A number of synthetic remedies are as gastroprotective
agents and hence control ulcer including antacids, H2
receptor blockers, proton pump inhibitors or
antihistamines [46, 47]. However, at present, these drugs
are coming across several limitations in terms of patient
safety issues involving confined efficacy against gastric
diseases and severe side effects [47-49]. Therefore, natural
gastroprotective agents from plant origin are being
significantly considered due to their collection from
convenient sources, less side-effects and cost
effectiveness. Various medicinal plants and crude drugs
are currently gaining global attention for their
pharmacological effect against gastric ulcer and other GI
disorders [47, 50]. Former animal and in vitro studies have
exhibited protective and curative effect of crude extract of
Bacopa monnieri against gastric ulcer [51-54].
At present, there is limited literature available to support
the antiulcerogenic effect of Bangladeshi species of
Bacopa monnieri L. leaf or whole plant extract using
various solvents. Therefore, the present study has been
designed to come up with scientific evidence for the use of
B. monnierias traditional ulcer healer by investigating the
antiulcerogenic activity of different extracts of Bacopa
monnieri (Linn.) on ethanol-induced gastric injury in
Swiss Albino mice.
2. Materials and methods
2.1 Chemicals and reagents
Omeprazole (API) was obtained fromthe pharmaceutical
industry Eskayef Bangladesh Ltd. Ethanol, carbon
tetrachloride, tween 80 and all other reagent materials
were purchased from Merck (Darmstadt, Germany) and
were of analytical grade.
2.2 Plant collection and extract preparation
Bacopa monnieri L., fresh plants, were collected on
January 2018 from Jahangirnagar University, Dhaka,
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Bangladesh. The identification of the plant material was
confirmed from Bangladesh National Herbarium, Dhaka
by comparing with their archived voucher specimen and
the register number is Bacopa monnieri - 46009. The
aerial parts of the plant were separated and washed
thoroughly. The plants were then kept in a warm dry place
(with adequate circulation of air), away from direct
sunlight, for two weeks to dry. Completely dried plant
material was ground into coarse powder by using a knife
mill (0.86µm). The obtained powdered plant material was
subjected to maceration process at room temperature with
water (500ml), 98% ethanol (500ml) and 99.9% carbon
tetrachloride (500ml). About 150gm of coarse powder was
soaked with each of the solvent for 21 days in a conical
flask covered with aluminium foil and consistent stirring
was done. The mixtures were then filtered using filter
paper (Whatman Filter paper No. 1) followed by
distillation process using rotary evaporator
(BuchiRotavapor R-200, Germany) at 78.370 C. The
aqueous extract was obtained by boiling at 600 C in a
water bath (JULABO GmbH, Germany). All three extracts
were air dried for three days in order to evaporate out any
remaining solvent. The residues of aqueous, ethanolic and
carbon tetrachloride extracts of Bacopa monnieri L. were
then collected, weighed and stored in an airtight container.
The extractive values of all three extracts were calculated
in percentage by using the following formula [55].
All drying, milling and extraction processes of crude plant
material were done from Pharmacognosy &
Phytochemistry Laboratory of Department of Pharmacy,
Primeasia University.
2.3 Phytochemical screening
Several phytochemical constituents like carbohydrates,
triterpenoids, alkaloids, glycosides, tannins, flavonoids,
essential oils and other similar secondary metabolites are
present in crude plant material. Hence, different
qualitative measures were taken to construct the
phytochemical profile of aqueous, ethanolic and carbon
tetrachloride extracts of Bacopa monnieri L.by using
standard protocols [56-58].
2.4 Experimental Animals
Swiss albino mice (Male) weighing 30-35 gm were
collected from the Department of Pharmacy,
Jahangirnagar University, Dhaka, Bangladesh. The
animals were housed in a separate animal room at the
Department of Pharmacy, Primeasia University where
they were kept in polypropylene cages in groups of six
mice per cage, maintaining a temperature of about 25 ±
20C with a 12-hour light-dark cycle. All animals were
allowed to acclimatize for one week with free access to
standard laboratory animal feed and water ad libitum prior
to the experiment. They were fasted for 24 hours with free
access to water only before the experiment [59]. All
surgical procedures were performed under isoflurane (5%
in 100% oxygen) anesthesia with an effort to minimize
pain. The Guide for the Care and Use of Laboratory
Animals (NIH publication No: 85-23, revised in 1985)
were followed for animal care and research protocols. The
experimental procedures were approved by the
Biomedical Research Center, University of Dhaka,
Bangladesh.
2.5 Induction of gastric ulcer
The animals were divided into nine groups (n = 6). The
normal group (Group I) received 0.2% tween 80 as vehicle
(0.5ml/100gm body weight) by oral gavage. The positive
control group (Group II) received oral dose of omeprazole
[60] in 0.2% tween 80 (v/v) as reference drug (20mg/kg of
body weight p.o.) and all the other test groups (Groups IV
- IX) received aqueous, ethanolic and carbon tetrachloride
extracts of B. monnieri at doses of 200 and 400 mg/kg
(b.w. p.o) [45] in 0.2% tween 80v/v (0.5ml/100gm body
weight) as a pretreatment. One hour later of the
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pretreatment, all the test groups (Groups II - IX) were
subjected to induction of gastric ulcer by receiving
absolute ethanol (0.5ml/100gm body weight) by oral
gavage. After one hour, the mice were euthanized
(sacrificed under anesthesia) and their stomachs were
dissected via cervical dislocation. The protocol of gastric
damage induced by ethanol has been taken from
previously established methods with a little modification
[61, 62]. The detail of treatment groups and experimental
protocol are given below:
Groups
Treatment
Group I
0.5 ml/100gm0.2% Tween 80, p.o.;
(Normal control)
Group II
0.5 ml/100gm0.2% Tween 80, p.o. + 5
ml/kg absolute ethanol p.o.
; (Ulcer
control)
Group III
20 mg/kg body weight omeprazole in
0.2% Tween 80, p.o. + 0.5
ml/100gmabsolute ethanol p.o.
(Positive control)
Group IV
200 mg/kg body weight aqueous
extract of Bacopa monnieri
in 0.2%
Tween 80, p.o + 0.5 ml/100gmabsolute
ethanol p.o.
Group V
400 mg/kg body weight aqueous
extract of Bacopa monnieri
in 0.2%
Tween 80, p.o + 0.5 ml/100gmabsolute
ethanol p.o.
Group VI
200 mg/kg body weight ethanolic
extract of Bacopa monnieri
in 0.2%
Tween 80
, p.o + 0.5 ml/100gm
absolute ethanol p.o.
Group VII
400 mg/kg body weight ethanolic
extract of Bacopa monnieri
in 0.2%
Tween 80, p.o + 0.5 ml/100gmabsolute
ethanol p.o.
Group VIII
200 mg/kg body weight carbon
tetrachloride extract ofBacopa
monnieri in 0.2% Tween 80, p.o + 0.5
ml/100gmabsolute ethanol p.o.
Group IX
400 mg/kg body weight carbon
tetrachloride extract of Bacopa
monnieri in 0.2% Tween 80, p.o + 0.5
ml/100gmabsolute ethanol p.o.
2.6 Evaluation of antiulcerogenic activity:
Determination of Ulcer Index (UI) and percentage of
ulcer protection
The stomachs of mice were opened along the greater
curvature followed by washing with physiological saline
(0.9% NaCl solution) to remove gastric contents and then
subjected to examination by a blinded pathologist for
gross macroscopic lesions under a magnifying glass. For
gross pathology, the degree of gastric mucosal damage
was evaluated based on a scoring system that involved
number and severity of gastric lesions as described by
previously established method [63]. The scoring for ulcer
was accounted as given below:
Condition
Scores
Normal stomach
0
Red coloration
0.5
Spot ulcer
1
Hemorrhagic streak
1.5
Ulcers
2
Perforations
3
The mean ulcer score for each group of animals was
calculated and expressed as ulcer index (UI) [64]. Later
on, the protection of ulcer was calculated and expressed in
percentage [65]. The formula for calculating UI and
percentage of ulcer protection are given below:
Calculation of ulcer index
UI = UN + US + UP × 10-1
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Where,
UI = ulcer index
UN = Average number of ulcer per animal group
US = Average of severity score
UP =Percentage of animal with ulcer
Calculation of % of ulcer protection
2.7 Histopathological examinations and microscopic
scoring of gastric damage
Histology of stomach of each group of animals was done
by isolating the organ and fixing it in freshly prepared
10% neutral buffered formalin at 4°C for 24 hours. The
tissue specimens were washed and dehydrated by alcohol.
Later on, they were cleared in xylene and embedded in
paraffin in hot air oven at a temperature of 560 C for 24
hours. Prior to histopathological examination under the
light microscope (LeicaMicrosystems, Germany), the
paraffin blocks were cut into tissue sections (5µm
thickness) and were subjected to staining with
hematoxylin and eosin (H&E) stains [66]. All
histopathologic procedures were conducted in Exim Bank
Hospital, Department of histopathology, Dhaka,
Bangladesh. In order to avoid any bias, a qualified
observer (who had been unaware of the identity of
samples) performed the evaluation of histopathological
tests.
Evaluation of microscopic damage score of gastric lesions
was done based on a scale of 0 – 14 according to the
criteria reported by Laine and Weinstein [67]. In brief,
1cm of gastric tissue segment was taken into account for
examination by observing epithelial cell loss (giving a
score of 0-3), edema in mucosal area (giving a score of 0-
4), hemorrhagic damage (giving a score of 0-4) and
presence of inflammatory & infiltrated cells (giving a
score of 0-3).
2.8 Statistical analysis
All experimental data were presented as mean ± SEM of
the mean (n = 6). Statistical analysis was done for the
comparison between control and treated groups by using
one-way analysis of variance (GraphPad Instat3).
Statistically significant differences of the P-value between
the groups were assessed by employing Dunnett’s test. A
value of P<0.05 was considered statistically significant.
3. Results
3.1 Extractive values of different extracts of Bacopa
monnieri
In the present study, three different extracts of Bacopa
monnieri L. were prepared using water, ethanol and
carbon tetrachloride solvent system. The extractive values
of the different solvent system were determined in
percentage as given in Table 1. The ethanolic extract
showed maximum extractive value (4%) while the
minimum extractive value was obtained by carbon
tetrachloride solvent system (2%). In the present
investigation, different colored extracts were obtained
(Table 1).
Table 1: Extractive value (in percentage) and color of
Bacopa monnieri L.
3.2 Qualitative phytochemical analysis
Preliminary phytochemical screening of Bacopa monnieri
L. showed presence of phytochemical constituents in the
different extracts (water, ethanolic and carbon
tetrachloride). Qualitative phytochemical analysis found
different important chemical compounds like alkaloids,
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flavonoids, phenols, tannins, steroids and terpenoids in all
three extracts as shown in Table 2.
Table 2: Analysis of phytochemical screening of Bacopa
monnieri L. in three different extracting solvents.
3.3 Effect of aqueous, ethanolic and carbon
tetrachloride extracts of Bacopa monnieri L. on
ethanol-induced gastric injury in mice
Evaluation of ulcer index and percentage protection
The severity of the gastric injury was explored based on
macroscopic damage scores done by gross evaluation of
stomach of mice. It was found that oral administration of
absolute ethanol (5 ml/kg p.o.) caused severe gastric
injury with high UI score when compared with normal
control group (treated with 0.2% tween 80).The values of
ulcer index (UI) and percentage protection obtained by
different animal groups treated with aqueous, ethanolic
and carbon tetrachloride extracts of B. monnieri are
demonstarted in Table 3. Pretreatment with both doses
(200mg/kg and 400mg/kg) of all three extracts (except
CTCEBM of 200mg/kg) showed significant result (**p<
0.01) for UI value when compared with ulcer control
(Group II). On the other hand, pretreatment with
CTCEBM (200mg/kg and 400mg/kg p.o.) exhibited
lowest UI score with the maximum ulcerative area (8%
and 12% ulcer protection). However, a moderate
protection against gastric injury was found by
pretreatment with aqueous extract of B. monnieri (20%
and 26% ulcer protection for the doses of 200mg/kg and
400mg/kg p.o. respectively). Interestingly, pretreatment
with EEBP (200mg/kg and 400mg/kg p.o.) showed lowest
score of gross gastric mucosal damage (3.13±0.38 and
2.75±0.32) and protected largest area of gastric damage
(78% and 81%) when compared with ulcer control. This
result was close to the score evidenced by the reference
anti-ulcer drug (omeprazole, 20mg/kg p.o.) showing 85%
ulcer protection (Figure 1). As demonstrated in Figure 1,
no significant difference was found in the UI values
between EEBP (200mg/kg and 400mg/kg p.o.) and
omeprazole (20mg/kg p.o.). Nevertheless, both aqueous
and carbon tetrachloride extracts of B. monnieri showed
significant difference (**p< 0.01) in UI values when
compared with the reference drug (omeprazole, 20mg/kg).
Table 3: Effect of different extracts of B. monnierion
changes in ulcer index and % of ulcer protection in mice
with ethanol-induced gastric injury.
Each value is Mean ± S.E.M (n=6). AEBM =Aqueous
Extract of B. monnieri, EEBM = ethanolic extract of B.
monnieri and CTCEBM = Carbon tetrachloride Extract of
B. monnieri. (*) indicates statistically significant
difference from respective group using one way analysis
of variance, followed by Dunnett’s multiple comparison
test (**p< 0.01). a = when compared with normal control,
b = when compared with ulcer control.
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Figure 1: Effect of B. monnieri extracts on ulcer index in
mice with ethanol-induced gastric injury. The values are
expressed as Mean ± S.E.M (n=6) where(*) indicates
statistically significant while (ƞ) indicates statistically
insignificant difference from respective group (Group III)
using one way analysis of variance, followed by
Dunnett’s multiple comparison test (**p< 0.01). Group III
= 20 mg/kg body weight omeprazole in 0.2% Tween 80,
p.o + 5 ml/kg absolute ethanol p.o.; Group IV = 200
mg/kg body weight aqueous extract of Bacopa monnieri in
0.2% Tween 80, p.o + 5 ml/kg absolute ethanol p.o.;
Group V = 400 mg/kg body weight aqueous extract of
Bacopa monnieri in 0.2% Tween 80, p.o + 5 ml/kg
absolute ethanol p.o.; Group VI = 200 mg/kg body
weight ethanolic extract of Bacopa monnieri in 0.2%
Tween 80, p.o + 5 ml/kg absolute ethanol p.o.; Group VII
= 400 mg/kg body weight ethanolic extract ofBacopa
monnieri in 0.2% Tween 80, p.o + 5 ml/kg absolute
ethanolp.o.; Group VIII = 200 mg/kg body weight carbon
tetrachloride extract of Bacopa monnieri in 0.2% Tween
80, p.o + 5 ml/kg absolute ethanol p.o.; Group IX = 400
mg/kg body weight carbon tetrachloride extract of Bacopa
monnieri in 0.2% Tween 80, p.o + 5 ml/kg absolute
ethanol p.o.
3.4 Effect of different extracts of B. monnieri on
histopathology of stomach of experimental mice
The present study further investigated whether the
ethanol-induced gastric models promoted histopathologic
damage when compared to normal control and the extracts
of Bacopa monnieri L. protected gastric damage when
compared to ulcer control. Samples of gastric sections
from each group displayed histopathologic changes as
shown in the Figure 2. The Figure 2A demonstrated a
section from the stomach of normal control mouse
received only 0.2% Tween 80 vehicle onlywithout
induction of ulcer. This section exhibits normal
architecture of mucosa (mu) with intact epithelial surface,
submucosa (sub) and muscularis (ml) layers. With respect
to the normal control the ulcer control (Figure 2B & C)
showed a diverse morphology of tissue that was
characterized by mucosal lesions with marked hemorrhage
(indicated by red arrows). In addition, the mucosa was
infiltrated by inflammatory cells (indicated by yellow
arrows) that also extended to the submucosa which also
displayed extensive edema. The histology of standard
group (Figure 2D) receiving omeprazole (20 mg/kg b.w.
p.o.) revealed attenuated morphological modifications,
diminished inflammatory cell invasion and mucosal
preservation. Pretreatment with aqueous extract of B.
monnieri (200 mg/kg and 400 mg/kg b.w. p.o.) failed to
preserve cellular morphology at higher extent as it showed
minor sign of hemorrhage (marked in red arrows), and
considerable loss of epithelial cells (marked in red square)
along with cellular infiltration (marked in yellow arrows)
and extensive edema in submucosal area (Figure 2E & F).
However, histology of B. monnieri extract of 400 mg/kg
exhibited slightly better morphology than the extract of
200 mg/kg in terms of the extent of cellular infiltration in
the area of submucosa (Figure 2F). In contrast,
administration of carbon tetrachloride extract of B.
monnieri (200 mg/kg and 400 mg/kg b.w. p.o.) could not
preserve cellular morphology at higher extent as minor
sign of hemorrhage (marked in red arrows) was found in
histopathological results along with considerable loss of
epithelial cells (marked in red square) and cellular
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infiltration (marked in yellow arrows) (Figure 2G & H). Interestingly, pretreatment with ethanolic extract of B. monnieri
(200 mg/kg and 400 mg/kg b.w. p.o.) preserved the architecture of the gastric wall (Figure 2I & J).
Based on the results of mean microscopic damage score, as demonstrated in Figure 3, the ethanol control mice exhibited
highest score (12 out of 14) showing severe mucosal injury. Significant difference (**p< 0.01) was found between ulcer
control group and all other treatment groups. Pre-treated with aqueous and carbon tetrachloride extracts of B. monnieri
before being given ethanol (0.5ml/kg b.w. p.o.) had shown very high score indicating significant difference (**p< 0.01)
when compared with group pre-treated by omeprazole (20 mg/kg b.w. p.o.). However, there were no significant
differences in microscopic damage scores between ethanolic extracts of 200 and 400 mg/kg of B. monnieri (3.5 and 3 out
of 14 respectively) and the reference drug (omeprazole) (2 out of 14). This significantly reduced areas of gastric ulcer
formation showing flattening of gastric mucosal folds.
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Figure 2: Histopathological changes in the stomach
representing photomicrographs of sections from gastric
walls of different groups of ulcer induced experimental
animals. A: Group I = Normal control; B & C: Group II
= Ulcer control; D: Group III = 20 mg/kg body weight
omeprazole in 0.2% Tween 80, p.o + 5 ml/kg absolute
ethanol p.o.; E:Group IV = 200 mg/kg body weight
aqueous extract of Bacopa monnieri in 0.2% Tween 80,
p.o + 5 ml/kg absolute ethanol p.o.; F:Group V = 400
mg/kg body weight aqueous extract of Bacopa monnieri in
0.2% Tween 80, p.o + 5 ml/kg absolute ethanol p.o.;
G:Group VI = 200 mg/kg body weight ethanolic extract
of Bacopa monnieri in 0.2% Tween 80, p.o + 5 ml/kg
absolute ethanol p.o.; H:Group VII = 400 mg/kg body
Figure 3: Microscopic damage scores (0-14) expressed as
Mean ± S.E.M.; n=6.Normal control = 5 ml/kg 0.2%
Tween 80, p.o.; Ulcer Control = 5 ml/kg 0.2% Tween 80,
p.o. + 5 ml/kg absolute ethanol p.o. AEBM =Aqueous
Extract of B. monnieri, EEBM = ethanolic extract of B.
monnieri and CTCEBM = Carbon tetrachloride Extract of
B. monnieri. (*) indicates statistically significant while (ƞ)
indicates statistically insignificant difference from
respective groups using one way analysis of variance,
followed by Dunnett’s multiple comparison test (**p<
0.01). a = when compared with ulcer control, b = when
compared with omeprazole.
4. Discussion
A wide variety of herbal drugs have been approved by
Food and Drug Administration (FDA) that are currently
available in global market. These herbal remedies have
been recognized for their effectiveness against several
ailments due to the presence of significant phytochemical
constituents [68-70]. The present investigation involved
phytochemical analysis of three different extracts
including water, ethanol and carbon tetrachloride of
Bacopa monnieri L. Extract of different solvent medium
exhibited variation in availability of phytochemical
constituents. Both aqueous and carbon tetra chloride
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extracts of Bacopamonnieri L. showed the presence of
useful phytochemical constituents including alkaloids,
flavonoids, phenols, steroids, glycosides, tannins and
terpenoids. In contrast, all of these phytochemical
constituents are present in the ethanolic extract of the
plant except for glycosides. However, saponin was not
found in the extract of any of the solvent medium. Such
observation regarding phytochemical screening of
aqueous and ethanolic extracts of Bacopa monnieri L. was
observed in previous study by Singh et al., 2012 [71].
Gastric ulcer has been recognized as global concern for
many years and is characterized as inflammation in gastric
mucosa. Medicinal plants containing flavonoids and
phenols are significantly used as protective agents against
ulcers [62, 72]. Our present study highlights the protective
activity of various extracts of Bacopa monnieri L. against
gastric ulcer. The study revealed variation in anti-
ulcerogenic effect of three different extracts (aqueous,
ethanolic and carbon tetrachloride) of B. monnieri against
ethanol-induced gastric injury in mice. Due to presence of
flavonoids in all three extracts of the plant, moderate to
extensive protection against gastric ulcer was observed.
Based on previous report, ethanol induced gastric lesions
cause mucosal damage by producing considerable
inflammatory cell infiltration and tissue injury [73-75].
Such models have been widely used for the purpose of
evaluation of gastroprotective activity [76-80]. As per our
histopathological results, exposure of ethanol to gastric
mucosa inflicted gastric injury by disruption of mucosal
and submucosal layers. In addition, the histopathological
results and microscopic damage scores of the treated
animals with aqueous and carbon tetrachloride extracts of
B. monnieri exhibited moderate to poor protection against
gastric ulcers when compared with treatment with
standard drug (Omeprazole). On the other hand, the
ethanolic extract of B. monnieri (200 & 400 mg/kg b.w.)
potentially regulated the inflammatory response that
correspond to omeprazole (20 mg/kg b.w.) as evidenced
by reduced ulcer index and microscopic score along with
the results of histopathological evaluation. Although all
three extracts contain flavonoids and phenols, the present
outcome of our study suggests that the presence of high
flavonoid and phenolic contents may favor the ethanolic
extract of B. monnieri to possess significant protective
effect against ulcer when compared with other two
extracts.
5. Conclusion
In conclusion, the present study evidently showed anti-
ulcerogenic activity of ethanolic extract of Bacopa
monnieri L. against ethanol-induced gastric ulcer in mice
model. This extract exhibited dose-dependent therapeutic
effect. As a result, high dose (400 mg/kg b.w) of ethanolic
extract of B. monnieri showed significant protection
against gastric ulcer. At this dose, the therapeutic efficacy
of the extract was observed to be same as omeprazole (20
mg/kg b.w.). Hence, with respect to our present analysis, it
can be stated that ethanol could be a suitable medium to
obtain Bacopa monnieri L. extract which can be
considered as a promising curative for the treatment of
gastric ulcer.
Ethics approval
All experimental procedures were approved by the
Biomedical Research Center, University of Dhaka,
Bangladesh.
Acknowledgments
Laboratory support from the Department of Pharmacy,
Primeasia University, Dhaka, Bangladesh and
Phytochemical Laboratory of University of Dhaka, Dhaka,
Bangladesh is gratefully acknowledged.
Abbreviations
B. monnieri L. = Bacopa monnieri Linn.
UI = Ulcer index
AEBM =Aqueous Extract of B. monnieri
EEBM = ethanolic extract of B. monnieri
Rubaba Karim, et al. International Journal of Medical Sciences and Innovative Research (IJMSIR)
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CTCEBM = Carbon tetrachloride Extract of B. monnieri.
Competing interest
The authors declare that they have no competing interests.
Funding
The present research was conducted for one year. The cost
of research was high and we did not have grant or
received any departmental funding.
Consent for publication
We confirm that this manuscript has not been published
elsewhere and is not under consideration by another
journal. All authors have approved the manuscript and
agreed with submission to International Journal of
Medical Science and Innovative Research (IJMSIR).
Availability of data and materials
All data generated or analysed during this study are
included in this published article.
Authors' contributions
Rubaba Karim made substantial contributions to
conception design and conduction of research. Sabiha
Akter, Najia Sultana Shuchi, Mst. Maliha Tabachasum
Madhuri, and Md. Nazmul Hossain performed all of the
experiments in the laboratory. Data collection, statistical
analysis and interpretation were done by Rubaba Karim
and Ashfia Fatima Khan. Article was written by Rubaba
Karim. Critical revision of the article was done by Ashfia
Fatima Khan. All Authors read and approved the final
manuscripts.
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